The present invention relates to the field of recombinant deoxyribonucleic acid (DNA) technology. Specifically, the invention relates to identification of the genes responsible for poly-beta-hydroxybutyrate biosynthesis pathway from Streptomyces aureofaciens NRRL 2209, creation of a plasmid vector carrying the said gene and expression of this gene in Escherichia coli designated as NCIM 5128 and bearing ATCC Accession No. PTA 1579, which is used for synthesis of polyhydroxybutyrate in recoverable amounts of at least 60% of dry bacterial cell mass.
Lemoigne in 1926 discovered the presence of PHB (poly-beta-hydroxybutyrate) in Bacillus. This has been reported to be present in a multitude of other bacterial genera, including Azotobacter, Alcaligenes, Psuedonomas, Rhizobium, Chromatium, Acinetobacter, Rhodospirillum and some species of cyanobacteria. It is also reported to be present in certain actinomycetes in very minute quantities. PHB is synthesized and stored by these microorganisms essentially as an energy source under stress conditions. PHB, a homopolymer of D-(xe2x88x92)-3-hydroxybutyrate, has properties comparable to synthetic polymers like polypropylene. PHB is commercially produced by fermentation technology using Alcaligenes eutrophus (Ralstonia eutropha) and is marketed under the brand name Biopol. In the present global environment awareness, as against synthetic polymers which are persistent by nature, PHB is bestowed with the property of biodegradability. Besides being used in packaging industry, PHB has also been used as a source of chiral centers for the organic synthesis of certain antibiotics, in drug delivery and bone replacement applications. The biosynthesis of PHB has been studied extensively in Alcaligenes eutrophus, Rhodospirullum rubrum, Pseudomonas species and Azotobacter beijerinckii. xcex2-ketothiolase, the first enzyme in the pathway and coded for by the phaA gene, first catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is then reduced to D-(xe2x88x92)-3-hydroxybutyryl-CoA by NADPH dependent acetoacetyl-CoA reductase which is coded for by phaB gene. D-(xe2x88x92)-3-hydroxybutyryl-CoA monomer is then polymerized to PHB by PHB synthase coded for by the phaC gene. PHB in the bacterial cell accumulates as cytoplasmic inclusions when growth of the bacteria in culture is limited by a nutrient other than a carbon source. It may be oxygen deprivation, nitrogen deprivation, phosphate limitation, sulfate limitation and magnesium limitation. Once the limiting conditions are relaxed, PHB is metabolized down to preinduction levels. It has been shown that both xcex2-ketothiolase and acetoacetyl CoA reductase activities increase in response to PHB-stimulating limitation conditions.
Some of the U.S. Patents covering production and extraction of PHB from microorganisms include the following: U.S. Pat. No. 4,786,598 to Lafferty et al. discloses a two-stage fermentation process where PHB is produced using Alcaligenes latus, U.S. Pat. No. 4,705,604 to Vanlautem et al. discloses using 1,2 dichloroethane to simultaneously remove water from the bacterial suspension by azeotropic distillation and extract PHB from the cells, U.S. Pat. No. 4,477,654 to Holmes et al discloses limiting the nitrogen nutrient source to microbiologically accumulate 3-hydroxybutyrate polymers, U.S. Pat. No. 4,433,053 discloses a fermenting process for PHB accumulation using A. eutrophus where a nutrient required for growth is limited, U.S. Pat. No. 4,336,334 to Powell et al. shows a microbiological process for producing PHB using Methylobacterium organophilum, U.S. Pat. No. 4,358,583 to Walker et al. discloses extracting PHB by first flocculating the cells by heat or pH treatment then extracting with a suitable solvent, U.S. Pat. No. 4,138,291 to Lafferty discloses bacterial strains assimilating various carbon sources and converting them to PHB, U.S. Pat. No. 5,518,907 to Dennis discloses Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway, U.S. Pat. No. 5,798,235 to Peoples, et. al., gene encoding bacterial acetoacetyl Co-A reductase and U.S. Pat. No. 5,650,555 to Somerville et. al. discloses transgenic plants producing polyhydroxyalkanoates. U.S. Pat. No. 5,512,456 to Dennis discloses method for the improved production and recovery of poly-beta-hydroxybutyrate from transformed Escherichia coli, U.S. Pat. No. 5,250,430 discloses Polyhydroxyalkanoate polymerase from Zoogloea ramigera. 
Among the prokaryotes, the actinomycetes constitute an important part of the microbial community responsible for the degradation and recycling of natural substrates. Accumulation of PHB has been reported from nine different strains of Streptomyces by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970). A possible role in thew biosynthesis of polyketide-derived phenolic metabolites such as actinorhodin or antimycin has been suggested by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970) and Packter and Flatman (Characterization of acetoacetyl-CoA reductase (3-oxoreductase) from Streptomyces coelicolor, its possible role in polyhydroxybutyrate biosynthesis. Biochem. Soc. Trans., 11:598-599, 1983). Streptomyces aureofaciens NRRL 2209 is a very poor accumulator of PHB and has been reported by Kannan and Rehacek (Formation of poly-beta-hydroxybutyrate by actinomycetes. Indian J. Biochem., 7:126-129, 1970) to accumulate PHB upto 1.10% of the dry cell mass. These bacteria are not as amenable as Escherichia coil to genetic manipulations and certainly are not as well characterized. Escherichia coli as a host cell has been exploited for producing molecules like the human growth hormone, insulin and interferon.
Thus, although polyhydroxybutyrate and other polyalkanoate biosynthesis genes have been isolated and characterized in various organisms such as Ralstonia eutropha (formerly known as Alcaligenes entrophus), there are several disadvantages in the use of these genes for production of polyalkanaoates on a large scale. For instance, when these genes are cloned into bacteria such as E. coli, the eventual production of alkanoate is not substantial. Further, there is no instance in the prior art wherein DNA fragments are isolated from actinomycetes and expressed in a heterogeneous host for production of polyhydroxybutyrate.
In order to make PHB production regulatable; a need exists for cloning of the PHB biosynthetic pathway from Streptomyces aureofaciens NRRL2209, its introduction and expression in Escherichia coli. 
The main object of the invention is to provide a method for cloning of genes coding for poly-beta-hydroxybutyrate biosynthesis pathway from Streptomyces aureofaciens NRRL2209 and expression thereof in Escherichia coli. 
Another object of the present invention is to identify the genomic sequences responsible for poly-beta-hydroxybutyrate biosynthesis pathway, from Streptomyces aureofaciens NRRL2209.
Yet another object of the present invention is to clone the genes responsible for poly-beta-hydroxybutyrate biosynthesis pathway, in a multicopy plasmid vector, thus creating a new vector which carries the nucleotide sequence responsible for the PHB synthesis pathway.
Yet another object of the present invention is to transform Escherichia coli with a multicopy plasmid vector for expression of the PHB biosynthesis pathway genes in the bacterial host.
Another object of the present invention is to produce PHB using transformed Escherichia coli in recoverable quantities.
Accordingly, the present invention provides a method for isolation of genes coding for poly-beta-hydroxybutyrate (PHB) biosynthesis pathway from Streptomyces aureofaciens NRRL2209, expressing the said genes in Escherichia coli and producing PHB using the transformed Escherichia coli to the extent of 60% or more of the cell weight.
Accordingly, the present invention provides a method for the production of poly-beta hydroxybutyrate (PHB) using recombinant Escherichia coli, said method comprising the steps of:
i) isolating the DNA sequence coding for the poly-beta-hydroxybutyrate (PHB) biosynthetic pathway, from Streptomyces aureofaciens NRRL2209,
ii) cloning the DNA sequence coding for PHB pathway into a plasmid vector pGEM-3Z to obtain a multicopy vector designated as pSa240,
iii) transforming Escherichia coli JM109 with the plasmid vectyor pSa240 to obtain recombinant Escherichia coli JM109 bearing accession No. PTA1579 and harbouring the gene responsible for production of PHB, and
iv) culturing recombinant Escherichia coli JM109 in a conventional medium containing glycerol and recovering poly-beta-hydroxybutyrate.
In an embodiment, the nucleic acid fragment coding for poly-beta-hydroxybutyrate synthesis pathway is a 4.826 Kb long fragment (SEQ ID NO: 1).
In another embodiment the nucleic acid fragment coding for PHB pathway is isolated from Streptomyces aurefaciens NRRL2209.
In yet another embodiment, the DNA sequence coding for PHB pathway is cloned into a multicopy plasmid vector named pGEM-3Z.
In another embodiment, the plasmid vector harbouring the gene coding for PHB pathway is pSa240.
In yet another embodiment, Escherichia coli JM109 is transformed with the multicopy plasmid vector pSa240 at a temperature in the range of 14xc2x0-18xc2x0 C. in the presence of T4 DNA ligase enzyme.
In another embodiment, the recombinant Escherichia coil JM109 is deposited with the American Type Culture Collection, USA and has received accession No. PTA1579. The deposit is made in compliance with the Budapest Treaty requirements.
In yet another embodiment, the transformed recombinant Escherichia coli JM109 when cultured in medium containing glycerol expresses the said biosynthetic pathway gene by producing poly-beta-hydroxybutyrate in recoverable quantities of at least about 60% of the dry cell mass of the Escherichia coli JM109 bacterial host.
The Escherichia coli JM109 bacterial host has been deposited with ATCC and bears Accession Number PTA No. 1579.
The transformed and genetically modified strain Escherichia coli JM109 harbors the plasmid pSa240 expresses gene for ampicillin resistance and the polyhydroxybutyrate biosynthetic operon obtained from Streptomyces aureofaciens NRRL2209. The original Escherichia coli JM109 strain was procured from ATCC and bears accession number 53323.
As said earlier, polyhydroxybutyrate and other polyhydroxyalkanoate biosynthesis genes have been isolated, characterized and patented from organism like Ralstonia eutropha (formerly known as Alcaligenes eutrophus), Rhodospirillum rubrum, Zoogloea ramigera. The Applicants have now have isolated, cloned, sequenced and characterized a 4.826 kb Sau3A I restriction endonuclease fragment (SEQ ID NO: 1) from the genomic DNA of Streptomyces aureofaciens NRRL2209 and this fragment carries all the genetic information required for the synthesis of polyhydroxybutyrate upon it being used to transform Escherichia coli JM109. This is the first instance of its kind where DNA fragment isolated from a member of actinomycetes has been expressed in a heterologous host for the production of polyhydroxybutyrate. The transformed Escherichia coli harboring the 4.826 kb Sau3A I restriction endonuclease fragment preferentially uses glycerol as a carbon source for the synthesis of polyhydroxybutyrate. The putative polyhydroxybutyrate biosynthesis genes that the applicants have isolated does not show significant sequence similarity at amino acid level with any of the reported polyhydroxybutyrate biosynthesis gene sequences. While Streptomyces aureofaciens NRRL2209 accumulates only about 1% polyhydroxybutyrate, the DNA fragment that the applicants have isolated from this organism and introduced into Escherichia coli JM 109 supports production and accumulation of polyhydroxybutyrate to the extent of 60% of dry cell mass of this heterologous host. The novelty of the patent is the isolation, cloning, sequencing and heterologous statement of a 4.826 kb DNA sequence (SEQ ID NO: 1) from Streptomyces aureofaciens NRRL2209 for the production of polyhydroxybutyrate.
The novelty of the invention resides in isolation, cloning, sequencing and characterization of a 4.826 kb Sau3A I restriction fragment from the genomic DNA of Streptomyces aureofaciens NRRL 2209. The Sau3A I DNA fragment harbors genes responsible for the synthesis of polyhydroxybutyrate. This Sau3A I DNA fragment when cloned and introduced into Escherichia coli JM109 as plasmid vector pSa240 supports the synthesis of polyhydroxybutyrate to the extent of at least 60% dry mass of the bacterial cell. The recombinant Escherichia coli JM109 (ATCC PTA-1579) utilizes glycerol as a carbon source for the synthesis of polyhydroxybutyrate.
However, with the use of other carbon sources individually or in combination it may be possible to order the synthesis of the other homo- or co-polymers of hydroxyalkanoates.